This application is a continuation of U.S. Ser. No. 09/156,809 filed Sep. 18, 1998, now U.S. Pat. No. 6,316,262, which issues Nov. 13, 2001, which claims priority to U.S. Ser. No. 60/059,373 filed Sep. 19, 1997, entitled Biological Systems for the Manufacture of Polyhydroxyalkanoate Polymers containing 4-Hydroxyacids by Gjalt W. Huisman, Frank A. Skraly, David P. Martin, and Oliver P. Peoples.
Poly [(R)-3-hydroxyalkanoates] (PHAs) are biodegradable and biocompatible thermoplastic materials, produced from renewable resources, with a broad range of industrial and biomedical applications (Williams and Peoples, 1996, CHEMTECH 26, 38-44). In recent years, what was viewed as a single polymer, poly-xcex2-hydroxybutyrate (PHB), has evolved into a broad class of polyesters with different monomer compositions and a wide range of physical properties. To date around one hundred different monomers have been incorporated into the PHA polymers (Steinbxc3xcchel and Valentin, 1995, FEMS Microbiol. Lett. 128; 219-228). It has been useful to broadly divide the PHAs into two groups according to the length of their side chains and their pathways for biosynthesis. Those with short side chains, such as polyhydroxybutyrate (PHB), a homopolymer of R-3-hydroxybutyric acid units,
xe2x80x94OCR1R2(CR3R4)nCOxe2x80x94
where: n is 0 or an integer and R1, R2, R3, and R4 are each selected from saturated and unsaturated hydrocarbon radicals; hal- and hydroxy-substituted radicals; hydroxy radicals; halogen radicals; nitrogen-substituted radicals; oxygen-substitued radicals; and hydrogen atoms,
are crystalline thermoplastics, whereas PHAs with long side chains are more elastomeric. The former have been known for about seventy years (Lemoigne and Roukhelman, 1925), whereas the latter materials were first identified in the early 1980""s (deSmet et al., 1983, J. Bacteriol., 154; 870-878). Before this designation, however, PHAs of microbial origin containing both (R)-3-hydroxybutyric acid and one or more long side chain hydroxyacid units containing from five to sixteen carbon atoms had been identified (Steinbxc3xcchel and Wiese, 1992, Appl. Microbiol. Biotechnol. 37: 691-697; Valentin et al., 1992, Appl. Microbiol. Biotechnol. 36: 507-514; Valentin et al., 1994, Appl. Microbiol. Biotechnol. 40: 710-716; Lee et al., 1995, Appl. Microbiol. Biotechnol. 42: 901-909; Kato et al., 1996, Appl. Microbiol. Biotechnol. 45: 363-370; Abe et al., 1994, Int. J. Biol. Macromol. 16: 115-119; Valentin et al., 1996, Appl. Microbiol. Biotechnol. 46: 261-267; U.S. Pat. No. 4,876,331). A combination of the two biosynthetic pathways probably provide the hydroxyacid monomers. These latter copolymers can be referred to as PHB-co-HX. Useful examples of specific two-component copolymers include PHB-co-3-hydroxyhexanoate (Brandl et al., 1989, Int. J. Biol. Macromol. 11; 49-55; Amos and McInerey, 1991, Arch. Microbiol. 155: 103-106; Shiotani et al., 1994, U.S. Pat. No. 5,292,860). Chemical synthetic methods have also been used to prepare racemic PHB copolymers of this type for applications testing (WO 95/20614, WO 95/20615 and WO 96/20621).
Numerous microorganisms have the ability to accumulate intracellular reserves of PHA polymers. Since polyhydroxyalkanoates are natural thermoplastic polyesters, the majority of their applications are as replacements for petrochemical polymers currently in use for packaging and coating applications. The extensive range of physical properties of the PHA family of polymers, in addition to the broadening of performance obtainable by compounding and blending as traditionally performed in the polymer industry, provides a corresponding broad range of potential end-use applications. The PHAs can be produced in a wide variety of types depending on the hydroxyacid monomer composition (Steinbxc3xcchel and Valentin, 1995, FEMS Microbiol. Lett. 128: 219-228). This wide range of polymer compositions reflects an equally wide range of polymer physical properties including: a range of melting temperatures from 40xc2x0 C.-180xc2x0 C., glass transition temperatures from xe2x88x9235 to 5xc2x0 C., degrees of crystallinity of 0% to 80% coupled with the ability to control the rate of crystallization and elongation to break of 5 to 500%. Poly(3-hydroxybutyrate), for example, has characteristics similar to those of polypropylene while poly(3-hydroxyoctanoate) (a copolymer of (R)-3-hydroxyoctanoate and (R)-3-hydroxyhexanoate) types behave more as elastomers and PHAs with longer side chains giving behavior closer to waxes. The PHAs can also be plasticized and blended with other polymers or agents. One particularly useful form is as a latex of PHA in water.
The monomer compositions also affect solubility in organic solvents allowing for a choice of a wide range of solvents. Copolymers of (R)-3-hydroxybutyrate and other hydroxyacid comonomers have significantly different solubility characteristics from those of the PHB homopolymer.
To date, PHAs have seen limited commercial availability with only the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) being available in significant quantities. This copolymer has been produced by fermentation of the bacterium Ralstonia eutropha (formerly Alcaligenes eutrophus). Fermentation processes for other PHAs have been developed (Williams and Peoples, 1996, CHEMTECH 26: 38-44). Plant crops are also being genetically engineered to produce these polymers, and offer a cost structure in line with the vegetable oils and direct price competitiveness with petroleum based polymers (Williams and Peoples 1996, CHEMTECH 26: 38-44). More traditional polymer synthesis approaches have also been examined, including direct condensation and ring-opening polymerization of the corresponding lactones (Jesudason and Marchessault, 1994, Macromolecules 27: 2595-2602, U.S. Pat. Nos. 5,286,842; 5,563,239; 5,516,883; 5,461,139; Canadian patent application 2,006,508).
Synthesis of PHA polymers containing the monomer 4-hydroxybutyrate (PHB4HB, Doi, Y. 1995, Macromol. Symp. 98, 585-599) or 4-hydroxyvalerate and 4-hydroxyhexanoate containing PHA polyesters have been described (Valentin et al., 1992, Appl. Microbiol. Biotechnol. 36: 507-514 and Valentin et al., 1994, Appl. Microbiol. Biotechnol. 40: 710-716). These polyesters have been manufactured using methods similar to that originally described for PHBV in which the microorganisms are fed a relatively expensive non-carbohydrate feedstock in order to force the incorporation of the monomer into the PHA polyester. For example, production of PHB4HB has been accomplished by feeding glucose and 4-hydroxybutyrate or substrate that is converted to 4-hydroxybutyrate to A. eutrophus (Kunioka, M., Nakamura, Y., and Doi, Y. 1988, Polym. Commun. 29: 174; Doi, Y., Segawa, A. and Kunioka, M. 1990, Int. J. Biol. Macromo. 12: 106; Nakamura, S., Doi, Y. and Scandola, M. 1992, Macromolecules 25: 423) A. latus (Hiramitsu, M., Koyama, N. and Doi, Y. 1993, Biotechnol. Lett. 15: 461), Pseudomonas acidovorans (Kimura, H., Yoshida, Y. and Doi, Y. 1992, Biotechnol. Lett. 14: 445) and Comomonas acidovorans (Saito, Y. and Doi, Y., 1994, Int. J. Biol. Macromol. 16: 18). Substrates that are converted to 4-hydroxybutyrate are 1,4-butanediol, 1,6-hexanediol, 1,8-octanediol, 1,10-decanediol, 1,12-dodecanediol and 1,4-butyrolactone. The PHB4HB copolymers can be produced with a range of monomer compositions which again provides a range of polymer properties. In particular as the amount of 4HB increases above 10 wt. %, the melting temperature (Tm) decreases below 130xc2x0 C. and the elongation to break increases above 400% (Saito, Y., Nakamura, S., Hiramitsu, M. and Doi, Y., 1996, Polym. Int. 39: 169).
The formation of 4HB containing polymers has also been studied with recombinant strains in studies aimed at improved PHB-4HB formation in Ralstonia eutropha or E. coli. Mutants of R. eutropha H16 were selected that cannot use 4-hydroxybutyrate as a carbon source. When such mutants were tested for copolymer formation, up to 84% 4HB was incorporated into the accumulated PHA (Kitamura S and Y. Doi, 1994. in Biodegradable Plastics and Polyesters, 12, p. 373-378). By introducing additional copies of the phb genes, the accumulation of PHB-4HB was enhanced (Lee, Y.-H., Park, J.-S. and Huh, T.-L. 1997, Biotechnol. Lett. 19: 771-774).
It is desirable to develop more cost effective ways of producing PHAs containing 4HB by biological systems. Several factors are critical for economic production of PHA: substrate costs, fermentation time, and efficiency of downstream-processing. A general characteristic of the above described bacteria is that their growth rate is low, they are often difficult to break open and their amenity to genetic engineering is limited. Therefore, processes have been developed that improve the economics of PHA production by using transgenic organisms. Formation of PHB4HB was achieved in E. coli using the 4-hydroxybutyrate pathway from C. kluyveri (Hein, S., Sxc3x6hling, B., Gottschalk, G., and Steinbxc3xcchel, A. 1997. FEMS Microbiol. Lett. 153: 411-418). In these studies both the 4-hydroxybutyryl-CoA transferase and PHA synthase were plasmid encoded. Subsequent work showed that the 4-hydroxybutyrate pathway from C. kluyveri supports formation of PHB-4HB in E. coli up to 50% of the cell dry weight from glucose as sole carbon source, and where 2.8% of the monomers is 4HB. The 4HB monomer in these strains is most likely derived from succinate, an intermediate of the TCA cycle (Valentin, H. E. and Dennis, D. 1997. J. Biotechnol. 58: 33-38). These studies were based on Escherichia coli as recombinant production organisms and PHA biosynthetic genes from PHA producers such as R eutropha. 
It is an object of the present invention to provide recombinant processes whereby additional genes can be introduced in transgenic PHB producers to create new strains that synthesize monomers, such as 4HB, for alternative PHAs.
A further object of the present invention is to provide techniques and procedures to stably engineer transgenic organisms that synthesize PHAs containing 4-hydroxybutyrate either as sole constituent or as co-monomer.
It is also an object of the present invention to provide screening systems for new 4-hydroxybutyryl CoA transferase encoding genes.
It is another object of the present invention to provide techniques and procedures to engineer new pathways in biological systems for the endogenous synthesis of alternative PHA monomers.
Improved production processes for 4HB containing PHAs using transgenic strains have been developed. Transgenic E. coli strains are described in which the required phb genes have been integrated on the chromosome. Additional genes for the synthesis of the 4HB monomer are also integrated on the chromosome. The latter genes can be derived from a broad range of organisms which carry a 4-hydroxybutyryl-CoA transferase and be identified by screening for this activity in the engineered E. coli strains described here. In addition, an endogenous E. coli activity is disclosed that can be further improved for the purpose of 4HB-CoA transferase activity. New pathways are also disclosed for the supply of intermediates of 4HB biosynthetic pathways such as xcex1-ketoglutarate and xcex3-aminobutyrate. The diversity of these pathways is important for the successful production of 4HB containing PHAs from cheap carbon sources such as sugars and fatty acids.